MX2010014026A - Novel adjuvant compositions. - Google Patents

Abstract

This invention relates to adjuvant formulations comprising various combinations of thterpenoids, sterols, immunomodulators, polymers, and Th2 stimulators; methods for making the adjuvant compositions; and the use of the adjuvant formulations in immunogenic and vaccine compositions with different antigens. This invention further relates to the use of the formulations in the treatment of animals.

Description

NOVELTY ADJUVANT COMPOSITIONS TECHNICAL BACKGROUND This invention relates generally to novel drugs for enhancing the immune response to be used in immunogenic and vaccine compositions, without toxic or undesirable side effects in the subject. This invention relates to methods of preparation and use of the immunogenic and vaccinal compounds.

TECHNICAL FIELD Bacterial, viral and parasitic infections and endured between human beings animals. They are a vaccine formulation. It can be done that S ace vaccines including an appropriate adjuvant in the composition.

There is also increasing interest in the use of vaccines to treat cancer in animals and in years. This therapeutic strategy to cancer treatment unites cancer patients with a tumor-specific tumor vaccine and an adjuvant. However, no vaccines against cancer of this nature that developing has been authorized by the regulatory authorities. Since vaccines reduce tumors, one measure is the effectiveness of anticancer drugs.

The term 'adjuvant' generally refers to a material that increases the humoral or cellular immune response. Adjuvants are used to achieve two objectives: rale rationing of the antigens from the site of injections, such as an adjuvant. In addition, the production of ethics and subunits is expensive. The addition of an adjuvant to the use of a lower dose of antigen to stimulate a similar unit, thus reducing the production cost of the vaccinal agent is combined with an adjuvant can effectively the effectiveness of some medicinal agents injected Many factors must be considered in the selection. An adjuvant should cause a relatively slow release and absorption of the antigen in an effective manner with minimal amounts of toxicity, allergies, irritations and other desirable in the host. To be desirable, a virucidal adjuvant, be biodegradable, be capable of creating a high immunity, be able to stimulate a cross protection with multiple antigens, be effective with multiple speci? C and be safe for the host ( For example, it does not provoke a vaccination reaction, however, the number of adjuvants that previous equisites is limited.

The choice of an adjuvant depends on the needs, whether it is to increase the magnitude or the function of the resp-bodies, an increase in the immune response mediated by the cell's immunity to mucous membranes or a reduction in the genome. A number of adjuvants have been submitted, however, proven to be fully adequate for all vaccines. The study that was reported in the literature was the adjuvant com und (FCA) that contains a water-in-oil emulsion and extr obacterium. Unfortunately, ACF is poorly tolerated and can cause uncontrolled inflammation. Since the discovery of the FCA years ago, attempts have been made to reduce the unwanted bodies of the adjuvants.

Some other materials that have been used as akin to cancer or allergic responses) or pharmaceutical properties, for example, rapid dispersion or poor control of the dispersion to the injection, or swelling of the material).

Synthesized oils and derivatives of adjuvants have been used because they show a relatively high dispersion, but may be undesirable since aromatic hydrocarbons, which may be carcinogenic, are often desired. Ad found that some of these substances can produce waste and can never be completely removed from the soil when they are selected and formulated in an appropriate manner so that they can be relatively safe and not t The saponins obtained from the bark of the suda /// aya saponaria tree have been used as adjuvants during some of Lacaille-Dubois, M and Wagner H. (A review of the biological activities of saponins.) Phytomedicine vol 2 pages bargo, is to stimulate a response directed against an antigen or ecíficos. The saponins have a high affinity for the cholesterol complex with the cholesterol that is found in the membranes, calling the lysis of the cell. It has also been shown that rosis at the site of injection and that it is difficult to formulate them into particulate uctures. When used in vaccines that contain live cover, saponins alter the viral envelope and thus viral inogens.

To overcome the hemolytic and virucidal properties d has combined with cholesterol and phospholipids, which form an ecological known as immunostimulatory complex (ISCOM) OM (ISCOMATRIX). See Ozel M., et.al .; J. Ultrastruc. and Mol 102, 240-248 (1989). ISCOMs, when combined with an antibody, induce a response of cytotoxic T lymphocytes ibar or, although they greatly reduce the hemolytic diseases of ominate, "DDA"), and avirdine. DDA is a lipophilic ternary compound (amine) with two 18-carbon methyl alkenyl chains attached to a quaternary ammonium molecule with a molecular weight of 631. Its use as an adjuvant was discovered (Immunol. page 369, 1966). It has been reported that potent immune responses mediated by cells, and have been shown to induce humoral immune responses, many studies showing the efficacy of DDA as well as protein antigens, haptens, tumors, viruses, pro teins. (See Korsholm, K S., et al., Immunology, vol 121, p 3, 2007.) Most studies have been conducted with an animal, while only a few have been done in animals such as chickens (See Katz, D., et al., FEMS Imm; robiol, Vol 7 (4): 303-313, 1993.), pigs and cattle. The DDA to induce a reaction of hypertension of you or delayed Dialkenilo or divinilglicol. Et CARBOPOL® has been used in a few, but its use as an adjuvant has not been demonstrated.

It has been shown that some adjuvants estimate Th2, with examples as N- (2-d-cilamino-b-D-glucopyranosyl) -N-octadecyldodecanelamide hydroacetate, Occupation with the trade name Bay R1005® when in tato, and aluminum. Bay R1005® combined with immunized vaccines or subunit vaccines cause greater prodrugs in mice exposed to viruses. Preclinical tests of animal species (pigs, sheep, horses) provided rpar- ters with respect to the production of antibodies. The increase in antibodies induced by the Bay R1005® depends on the specific antigen and not on the polyclonal stimulation.

Prior to this invention, no formulation would be the range of desirable characteristics that an ad uv BRIEF DESCRIPTION OF THE INVENTION This invention relates to novel ad nogenic and novel vaccine compositions. In particular, this invention relates to adjuvant formulations comprising nomodulatory stimulators, polymers, and Th2 stimulators. This also relates to immunogenic compositions and of vaccinating said adjuvant formulations and one or more antigens or to methods of preparing the adjuvant compositions.

In one embodiment, the adjuvant compositions include a combination of a saponin, a sterol, and an ernary compound. In one embodiment, the adjuvant combination comprises sterol, and DDA.

In another embodiment, the adjuvant compositions are prepared in one embodiment, comprising an adjuvant and a uniologically effective formulation of an antigen, wherein the formulation comprises a saponin, a sterol, a ternary compound, and a polymer by the process comprising a) preparing a composition of the antigen in a buffer b) adding the saponin to the composition of step a; c) adding the sterol to the composition of step b; d) adding the quaternary ammonium compound to the stage c, e) adding the polymer to the composition of step d.

In one embodiment of this process, saponin sterol is cholesterol, the quaternary ammonium compound is poly (aerypic acid).

In one modality, a vaccine is recom mended d) add the quaternary ammonium compound to the co to stage c, e) adding the polymer to the composition of step d, and f) adding the glycolipid to the composition of step e. In one embodiment of this process, the saponin sterol is cholesterol, the quaternary ammonium compound is mere poly (acrylic acid), and the glycolipid is Bay R1005®.

It has been found that the adjuvant compositions herein have surprising properties superior to those that would be expected from a combination surprisingly discovered that the virucidal property of sterol is eliminated in these adjuvant compositions. They are diluents for living modified viral antigens freeze-dried adjuvant positions described herein to be configured to elicit a response in. Applicants have discovered that these novel adjuvants are highly immunogenic when more than a number of antigens are combined. different from a great var ecies. They can be used with one or more viral antigens, baths, recombinant proteins, and synthetic peptides. Vaccine adjuvant compositions should not be used in therapeutic vaccines to treat cancer.

The present invention therefore provides compulvants, immunogens, and vaccines. There are also procedures for the manufacture of the compositions. It uses its use to treat a disease. It was also proposed to prepare a medicament for treating a disease subject, in particular against diseases that are depleted. Its use is also provided to prepare a med to prevent or reduce a disease in an area. diseases caused by canine coronavirus, to treat diseases caused by bovine rotavirus, and to treat diseases caused by canine influenza virus. It provides the use of adjuvants as a marker vaccine for the ationification of animals that have been vaccinated. CpG is also used to enhance the effects of adjuvants.

BRIEF DESCRIPTION OF THE INVENTION Figure 1 depicts a gel of a ioimmunoprecipitation showing the differences in the bodies between the NS2 / 3 proteins and the E2 proteins of the Po virus treated with PreZent A shows an antibody response to the NS2 / 3 proteins as against the proteins E2 while treated with QCDC CDCR demorado a The value indicated (for example, within the confidence interval of the mean) or within 10 percent of the indicated value, yor, unless it is used approximately to make time refunds in weeks in which "approximately 3 from 17 to 25 days, and from approximately 2 to approximately 4 from 10 to 40 days.

"Adjuvant" means any substance that puts a humoral or cellular immune against an antigen. The a generally used to achieve two objectives: slow the release from the site of injection, and stimulate the immune system "Alkyl" refers to saturated hydrocarbon radicals so branched.

"Amina" refers to a chemical compound that is arogen. Amines are a group of compounds that are substituted for the hydrocarbon atoms or the atoms of IgD, IgE, IgG, and IgM) based on the composition of the stanzas.

"Antigen" or "immunogen" refers to any sustained immune response. The term includes bacteria, killed, inactivated, attenuated or modified living sites. The gene also includes polynucleotides, polypeptides, mbinantes, synthetic peptides, protein extract, cellulose tumor cells), tissues, polysaccharides, or lipids, or their individual form or in any combination thereof. The gene also includes antibodies, such as for example, type antibody or its fragments, and synthetic peptide mimotopes which are an antigen or antigenic determinant (epitope).

"Bacterin" means a suspension of one or more drugs that can be used as a component of a vaccine or co-enzyme.

"Cellular immune response" or "immune cell response" is mediated by T lymphocytes or other leukocytes or the production of cytokines, chemokines, and molecules induced by T lymphocytes, leukocytes, or both.

"Cholesterol" refers to a chemical white crystalline substance of C27H45OH. It is a hydrocarbon alcohol cyclic as a lipid. It is insoluble in water, but soluble in an organic solvent.

"Delayed-type hypersensitivity (DTH)" is an inflammatory condition that develops from 24 to 72 hours after an antigen that the immune system recognizes as the type of immune response mainly involves the lymphocytes antibodies (which are produced by B lymphocytes).

"Dosage" refers to a vaccine or composition unmistaken to a subject. A "first dose" or "sensitization vaccine" "Emulsion" means a composition of two liquids in which small droplets of a liquid are continuously suspended from another liquid.

"Esters" refers to any of a class of co-acids that correspond to inorganic salts, which are formed by a condensation reaction in which a molecule of an acid ne to an alcohol molecule with elimination of a molecule d "Excipient" refers to any component of a non-antigen.

"Homogenization" refers to a process of multiple components, whether similar or not similar, in the form of an orme.

"Humoral immune response" refers to a dyad by antibodies.

"Hydrophobic" means insoluble in water, which is not an "immunologically protective amount" or "unologically effective amount" or "effective amount to produce a r unitary" of an antigen is an amount effective to induce a unicogenic r in the receptor. It may be for the purpose of diagnosis or other tests, or it may be appropriate to come with signs or symptoms of disease, which include adverse effects, caused by the infection with aggravation.It can be induced either humoral or cell-mediated immunity or both. The immunogenic response of an immunogenic position can be evaluated, for example, by evaluating antibody titers, proliferic assays, or directly through the signs and symptoms of control exposure to a wild-type strain, while The doctor conferred by a vaccine can be evaluated by measuring, by eduction of the clinical ones, as or em in mortality, m "Complex nmunoestimul "or ISCOM" refers to the specific structure that is formed when Quil A is combined with col olipidos.

"Immunostimulatory molecule" refers to a molecule was an immune response.

"Lipids" refers to any of a group of compounds, which include fats, oils, waxes, sterols and triglycerols insoluble in water but are soluble in organic solvents that are not oily to the touch and together with carbohydrates and structural proteins. main of living cells.

"Lipophilus" means that it shows marked lipid traits.

"Liposome" refers to a spherical particle micronized by a lipid bilayer that includes an aqueous compartment in medicine for delivering an anticancer drug to an enzyme vaccine including subcutaneous, intramuscular, transdermal, intraperitoneal, intraocular, and intravenous administration.

"Pharmaceutically acceptable" refers to substantially the medically informed criterion, they are suitable for use in tissues of subjects without toxicity, irritation, allergic response indilar, which corresponds to a reasonable relationship between good and effective for the use that is pretend "Reactogenic capacity" refers to the effects on a subject in response to the administration of an immunogen, or a vaccine composition. It can occur in administration, and is usually evaluated in terms of symptom development. These symptoms may include infl uence and abscess. It is also evaluated in terms of operation, and severity. A "low" reaction, for example, would suppose only "non-symmetric" or "steroid" can be detected or "steroids" refers to any of a group of compounds belonging to a biochemical class of lipids, soluble in solvents. Organic and slightly soluble steroids comprise a system of four condensed rings of cyclohexane (six-carbon) condensed plus a four-carbon (five carbons).

"Esteróles" refers to compounds in animals duced biologically from terpenoid precursors. Co rings structure of steroids, which have a hydro group bound to carbon-3. The hydrocarbon chain of its fatty acids varies in length, usually from 16 to 20 bt, and can be saturated or unsaturated. The sterols have one or more double bonds in the ring structure and the amount of substituents attached to the rings. The sterols and their ceilings are essentially insoluble in water.

"DOCT50" refers to "dose of tissue culture infection as that dilution of a virus necessary to infect the given one of inoculated cell cultures." Edits may be used to calculate DOCT5o, which includes the Arman-Karber procedure used in all this descriptive description of the Spearman-Karber procedure, see BW Mah gro, Virology Methods Manual, pages 25-46 (1996).

"Therapeutically effective amount" refers to an antigen or vaccine that would induce an immune response in receiving the antigen or vaccine that is adequate to prevent or re se or disease symptoms, including adverse effects of its complications, caused by infection with a po for example a virus or a bacterium. Immunity mediated by cells or both humoral and immunity can be induced. The immune response of an animal to a vaccine is used, of the particular antigen that is used, or of the state of being determined by one skilled in the art.

"Treat" refers to preventing a disorder, condition or in which the term is applied, or preventing or reducing one or more disorder, condition or disease.

"Treatment" refers to the act of "treating" as above.

"Triterpenoids" refers to a large class and natural organic molecules derived from isoprene-butadiene units of five carbons, which can be assembled and modified in form. Most are multi-cyclical structures that differ functional groups and in their skeletons of basic carbons can be found in all classes of living organisms.

"Vaccine" refers to a composition that includes one or is defined in the present document. The administration of the Components of the Compositions Triterpenoids and CpG Triterpenoids suitable for use in compulvants can come from many sources, whether synthetic synthetic derivatives, including but not limited to Quillaja s atina, ginseng extracts, mushrooms and a glucoside structurally similar to steroidal saponins. Thus, triteriates for use in the adjuvant compositions include saline and lanosterol. The amount of triterpenoids suitable for adjuvant compositions depends on the nature of the triterpe use. However, they are generally used in a range of about 1 to about 5,000 μg per dose. Ta n in an amount of about 1 μg to about approximately 5 μg to about 100 μg per dose, from about 30 μ9 to about 75 per day If a saponin is used, the compositions ad- minally contain an immunological saponin fraction in the bark of Quillaja saponaria. The saponin can be, for example, another preparation of purified or partially purifi ed saponins to be obtained commercially. Thus, purified extracts or mixtures or individual components can be used such as -17, QS-18, and QS-21. In one modality the Quil A has a pure 85%. In other modalities, Quil A has a purity of, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

ODNs with CpG are a class of recently described acotherapeutics characterized by a non-methylated CG dinucleotide in contexts immunomodulatory species similar to bacterial DNA. The cell surface can pick up these molecules with molecules. However, with a vehicle such as QCDC, for example, the combinations cited in this patent, the immunomodulation and capturing properties are significant.

The amount of CpG to be used in the compositions ad of the nature of the CpG that are used and of the species have. However, they are generally used in a maximum of 1 μg to about 20 mg per dose. Ta n in an amount of about 1 μ9 to about a dose, from about 1 μg to about 5 mg per 1 μg to about 4 mg per about 1 μg to about 3 mg per 100 μ9 per dose, and in an amount from about μ9 to about 75 μ9 per dose.

I loved you Suitable sterols for use in commissals include β-sitosterol, stigmasterol, ergosterol, ergoca sterol. These sterols are notorious in the art and can be cially. For example, cholesterol is described in The Me a Ed., Page 369. The amount of sterols suitable for adjuvant use depends on the nature of the sterol which, however, is generally used in an amount of approximately about 5,000 μ 9 per dose. . They are also used in an approximately 1 μg to approximately 4,000 g for approximately 1 μg to approximately 3,000 μg for Immunomodulators Adjuvant compositions can also include immunomodulatory agents such as, for example, brominated compounds (eg, DDA), and interleukins, interferons, or other materials can be purchased commercially. The suitable nomodulatory amount for use in the compositions depending on the nature of the immunomodulator used and its substance is generally used in an amount of approximately 5,000 μg per dose. They are also used in a maximum of 1 μg to about 4,000 μg per 1 μg to about 3,000 μg to about 1 μg to about 2,000 μg per dose from 1 μg to about 1,000 μg per dose. Ta in an amount of approximately 5 μg to aproximadament Polymers The adjuvant compositions may further include polymers such as, for example, DEAE Dextran, polyethylene glycol, and lico) and poly (methacrylic acid) (e.g., CARBOPOL®). Dich of being bought commercially. The amount of suitable polymers in the adjuvant compositions depends on the nature of the materials used. However, they are generally used at approximately 0.0001% by volume by volume, approximately 75% v / v. In other embodiments, they are used in a maximum of 0.001% v / v approximately 50% maximally 0.005% v / v approximately 25% approximately 0.01% v / v approximately 10% approximately 0.05% v / v approximately 2% v / v, approximately 0.1% v / goes approximately 0.75% v / v. In another m used in an amount of approximately 0.02 v / v a to roxim BOPOL® swell in water up to 1000 times its volume or times its original diameter forming a gel when exposed to pH higher than the pKa of the carboxylate group. At a super carboxylate pH, the carboxylate groups are ionized causing the negative charges, which adds to the swelling of the polymer.

Stimulants of Th2 The adjuvant compositions may also include Th2 stimulants such as, for example, Bay R1 inio. The amount of Th2 stimulants suitable for using adjuvant positions depends on the nature of the stimulant used. However, 0.01 g to about 10 mg per dose are generally used in a large amount. Aliments, are used in an amount of approximately 0.0 ximately 7.5 mg per dose, of approximately 0.1 morphs, is chemically stable in air and light at temperatures of in aqueous solvents at pH 2-12 at room temperature, amphiphilic écula that forms micelles in solution watery Antíqenos and diseases The adjuvant compositions may contain an antigens. The antigen can be any of a wide variety capable of producing a desired immune response. Although Quil A alone is viricidal, Quil A detoxifies cholesterol ion by forming helical micelles (See US Pat. No. 7, 122,191). It has been found that the adjuvants described herein are not virolytic or membranolytic. Thus, antigens that are used in adjuvant positions can be one or more of viruses (naïve, living modified), bacteria, parasites, nutes, polypeptides, which can be isolated from the organisms referred to herein.

Live, modified and attenuated live strains that do not occur in a subject have been isolated in a non-virulent manner or by using procedures well known in the art, including in a suitable cell line or exposure to ultraviolet-chemical light. Inactivated or killed viral strains are inactivated by methods known to the artisan, which include treatment with formalin, betapropriolacto binary neemine (BEI), sterilizing radiation, heat or other type processes.

Two or more antigens can be combined to produce a polyvalent position that can protect a subject against an age of diseases caused by pathogens. Actualm icant of commercial vaccines, as well as end-users, re Some examples of bacteria that can use iogens with adjuvant compositions include but are not inetobacter calcoaceticus, Acetobacter paseruianus, Acti uropneumoniae, Aeromonas hydrophila, Alicyclobacillus acido aeglobus fulgidus, Bacillus pumilus, Bacillus stearothermophilus Isma'ilis, Bacillus thermocatenulatus, Bordetella bronchiseptica, Bu acia, Burkholderia glumae, Campylobacter coli, Campylobac pylobacter jejuni, Campylobacter hyointestinalis, Chlamydia amydia trachomatis, Chlamydophila spp., Chromobacterium sipelothrix rhusiopathieae, Listeria monocytogenes, Ehrlichi Escherichia coli, Haemophilus influenzae, Haemophilus somnus , He s, Lawsonia intracellularis, Legionella pneumophilia, Morax cobactrium bovis, Mycoplasma hyopneumoniae, Mycoplasma? Sp. mycoides LC, Clostridium perfringens, Odoribacter d steurella Mannheimia haemol tica Pasteurella multocida Phot onella newport, Serratia marcescens, Spirlina platensis, Staphlus us, Staphylococcus epidermidis, Staphylococcus hyicus, Strep s, Streptomyces cinnamoneus, Streptococcus suis, Strep liates, Streptomyces scabies, Sulfolobus acidocaldarius , Syechoc or cholerae, Borrelia burgdorferi, Treponema denticola, Trum, Treponema phagedenis, Treponema refringens, Tr entii, Treponema palladium, and Leptospira species, such as th egens Leptospira canteóla, Leptospira grippotyposa, Leptospir ospira borgpetersenii hardjo-bovis, Leptospira borgpetersenii tno , Leptospira interrogans, Leptospira icterohaemorrhagiae, L ona, and Leptospira bratislava, and their combinations.

In the adjuvant compositions tactivates can be used as live attenuated viruses. Some examples of diseases to be used as antigens include, but are not limited to, her rivers, bovine herpesviruses, her canine esviruses, her esviruses and an equine infectious animal disease, feline immunodeficiency virus, feline fever (FeLV), Newcastle disease virus, sheep progressive virus, canine virus lung adenocarcinoma virus (CCV), pantropic CCV, bovine navirus respiratory coronavirus, feline calicivirus, feline enteric coronavirus, feline infectious tonitis, porcine epidemic diarrhea virus, swine haemagglutinating efalomyelitis, porcine parvovirus, Circoviru V) type I, type II PCV, reproductive and reproductive syndrome virus (PRRS), transmissible gastroenteritis virus, S coronaviruses of ephemeral bovine fever, rabies, Rotovirus, erynx virus, lentivirus, avian influenza , rhinovirus, swine influenza virus, canine influenza virus, feline influenza virus, iano virus, eastern equine encephalitis virus (EES), Venezuelan ae, West Nile virus, encephalitis virus, human immunodeficiency virus, papilloma virus. Some examples of parasites that can be used with adjuvant compositions include, but are not limited to, plasma, Fasciola hepatica (stave), Coccidia , Eimeria spp., Inum, Toxoplasma gondii, Giardia, Dirofilaria (worms c ylostoma (tapeworm), Trypanosoma spp., Leishmania spp., Trichomo ptosporidium parvum, Babesia, Schistosoma, Taenia, Stro aris, Trichinella, Sarcocystis, Hammondia, and Isopsora , ibinaciones. External parasites are also contemplated that, or without limitation, ticks, which include species of ipicephalus, Dermacentor, Amblyomma, Boophifus, Hyalo emaphysalis and their combinations.

The amount of antigen that is used to induce a unit will vary considerably depending on the subject antigen and the level of response desired and may be determined by one skilled in the art. For vaccines that ally, the therapeutically effective dose ranges from 104 DOCT50 to approximately 105 DOCT50, include For vaccines containing inactivated viruses, a pharmaceutically effective antigen is generally at most 100 relative units per dose, and is often ranging from about 1,000 to about 4,500 doses per dose, inclusive. In other embodiments, the pharmaceutically effective antigen is in an approximately 250 to about 4,000 relative usive units, from about 500 to about 3,000 tons per dose, inclusive, from about 750 to about 0 relative units per dose, inclusive, or approximately ximately 1, 500 relative units per dose, inclusive.

A therapeutically effective amount of antigen in inactivated virus can also be measured in terms of In one embodiment, a FeLV antigen was produced in the FL74-UCD-1 (ATCC number CRL-8012) which is consistent with the strain KT-FeLV-UCD -1 of FeLV antigen leukemia virus in a vaccine can be measured as the gp70 viral protein by me. A therapeutically effective amount FeLV, when measured by the amount of viral protein is generally in the range of about 350,000 ng / ml, inclusive. In another embodiment, from about 1,000 to about 300,000 ng / ml, including from 2,500 to about 250,000 ng / ml, including from 4,000 to about 220,000 ng / ml, including from about 5,000 to about 150,000 ng / ml, including about 10,000 ng / ml. / ml to approximately 100,000 ng / ml, i The number of cells for a bacterial antigen in a vaccine varies from approximately approximately 1 x106 to 5x10a CFU / dose, inclusive, or approximately 07 to 5x109 CFU / dose, inclusive.

The number of cells for a parasitic antigen in a vaccine varies in the range of approximately 1 xIO10 per dose, inclusive. In other embodiments, cells vary in the range of approximately 1 x 10 9 per dose, inclusive, or approximately 1 x 10 8 per dose, inclusive, or approximately 1 x 10 7 per dose, inclusive, or approximately 1 x 10 8 per dose, inclusive.

It is well known in the art that with adjuvants a substantially greater quantity of living modified or attenuated inactivated virus S is required to stimulate a serological composite level. However, it has been found surprising that the ad- uent compositions that are described in the one with wide utility need to be manufactured millions of doses to which these savings can be substantial.

Excipients Aqueous adjuvants provide certain reactions that are easy to formulate and administer and can induce severe reactions at the site of injection. No aqueous pools with an antigen tend to diffuse from the scion, are cleared by the liver of the subject and generate an undesired, non-specific r unitary. It has been found surprising that the aqueous adjuvant compositions described in the article remain at the site of the injection to etabolize, which occurs over a long period of time to provide a targeted immune response.

The oil, when added as a component of one or more components, has from 6 to 30 carbon atoms. Synthetically oiling or purifying from pe products can have a straight or branched chain structure. Fully saturated or having one or more double or triple bonds, non-metabolizable ites for use in the present invention, paraffin oil, and cycloparaffins, for example.

The term "oil" is also intended to include "oil G ?," that is, oil that is obtained in a similar manner by a distilled olate, but has a specific gravity of slightly white mineral ore.

The metabolizable oils include cos metabolic oils. The oil can be any vegetable oil, fish oil or synthetically prepared oil that can be metabolized by the subject to whom the adjuvant will be administered and who is not subject. The sources of vegetable oils include nuts, s Usually, the oil component of the present is present in an amount of 1% to 50% by volume; or in a 0% to 45%, or in an amount of 20% to 40%.

Other components of the compositions may be pharmaceutically acceptable, such as, for example, diluents, isotonic agents, buffering agents, preservatives, vasoconstrictive agents, bacterial agents, antifungal agents and the like. Typical vehicles, solvents and water include water, saline, dextrose, ethanol, ite, and the like. Representative isotonic agents include ico, dextrose, mannitol, sorbitol, lactose and the like. They are stabilized by gelatin, albumin and the like.

The surfactants are used to assist the stabilization selected to act as a carrier for the adjuvant. The surfactants suitable for use in the acetone oils which remain after removing the vegetable triglycerides by washing with acetone. Alternatively, lecitin nerse from various commercial sources. Other phospholipids ad yen phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, acid foolipin, and phosphatidylethanolamine. Phospholipids can be natural isolates or conventionally synthesized.

The non-natural synthetic surfactants suitable for this invention include non-ionic surfactants based on example sorbitan surfactants substituted with commercially available acids with the name of SPAN® or ARL res of polyethoxylated sorbitol fatty acids (TWEEN®), ie ethylene glycol fat from sources such as ULFOR® oil); polyethoxylated fatty acid (for example, onible acid with the name SIMULSOL M-53®), isoxylated / formaldehyde polymer (TYLOXAPOL®), alcohol ethers, dispersion grains, coatings, adjuvants, stabilizing agents, preservatives, antibacterial agents and antifungals, nicos, agents that delay adsorption and the like. They will be "acceptable" in the sense of being compatible with the components of the compositions and will not be harmful for the time being, the vehicles will be sterile and pyrogenic and will be selected in the administration mode to be used. It is well known in the art that the preferred formulations for the pharmaceutically acceptable comprising pharmaceutical compositions approved in the regulations to be followed by the US Department of Agriculture is (US) Department of Agriculture) or the US Food Agency. and Drug Administraron), or government agency in a different country of the United States. For the pharmaceutically acetained agent for sodium traduction, dextrose, mannitol, sorbitol and lactose among otilants include albumin, among others.

The compositions may also contain antibiotics including, for example, gentamicin, merthiolate or various classes of antibiotics or preservatives of which they are well known to those skilled in the art.

Composition Preparation Preparation of adjuvant formulations An ISCOM can be prepared by combining a sap ro! and a phospholipid. For example, an ISCOM may contain 5 weight of Quil A, 1% to 5% of cholesterol and phospholipids, and the rest ratio between saponin and sterol in the formulations will be in the order of 1: 100 weight per weight (p. / p) to 5: o of CARBOPOL® per part by weight of DDA. Still, at least 1 part by weight of CARBOPOL® per or DDA is used. The combination of CARBOPOL® and DDA forms a common functional group of tertiary amine of DDA immunofunctional carboxylic acid of the polymer. This allows the specific immune ones to target the antigen and the adjuvant and coadminister the antigen and adjuvant together at the optimal m centration to said cells.

The adjuvants described herein will not require any specific vehicle and be formulated aqueous or other pharmaceutically acceptable. In some cases, some of the described modalities will be presented in a quadrant, such as, for example, liposomes, microspheres or additional encapsulated genes. The antigen may be contiguous with the vesicles or contained on the outside of the m or spray-dried. The lyophilized compositions should be replaced before use to stabilize a solution, by saline or HEPES. Thus, the adjuvant compositions can be solid, semi-solid or liquid pharmaceutical form.

Adjuvants can be manufactured using techniques with technique. For example, saponin and cholesterol can mix adequate amount, followed by an extraction technique with d or liposomes or ISCOM. Saponin and cholesterol also coalesce into helical micelles as described in p. U.S. number 7,122,191.

Phosphat buffered saline or aqueous buffer medium can be used; The pH of the buffer can be either alkaline or slightly acidic. Accordingly, the pH may range from pH 6 to 8. A pH of about 7.3 is usual. The power of the buffer can be between P0 livantes. For example, adjuvants usually comprise approximately 1 pg to about 1000 pg, inclusive, of 1 ml. Similarly, antibiotics usually comprise approximately 1 pg to approximately 60 pg, inclusive, of a l.

The adjuvant formulations can be homogenized. The formulations are subjected to a primary process, usually passing one or more times through homogenizers. Any homogenizer can be used for this purpose, for example, Ross emulsifier (Ha, Gaulin homogenizer (Everett, MA), or Microfluidics (Newton, mode, the formulations are homogenized for three 00 rpm.) Microfluidization can be used by the fluidizer. commercial, such as the model number available in Microfluidics, Newton, Mass.; Gaulin Model 30CD The adjuvant compositions described in the umento can be either homogenized or microfluidific modality, an antigen is added to an appropriate buffer. and a saponin is slowly added to the antigenic solution, slowly add a sterol to the solution of antigen and saponin, the slow addition of a quaternary ammonium compound to the serum, saponin and sterol, and the resulting composition is then microfluidized. of the microfluidification, it is merely the microfluidized composition, depending on the com they are used, the in these stages for opration of the compositions.

Preparation of Immunogenic and Vacuum Compositions The adjuvant compositions described in the invention can be used for the manufacture of adjuvant compositions of the adjuvant compositions that are compliant depending on the characteristics desired position. For example, if a greater response is desired, the amount of the Th1 stimulator will be desired. Similarly, if greater Th2 response, the amount of the estim can be increased. A balanced response between JM immunogenic and vaccinal moieties can also be achieved by homogenisation as described above.

Administration and Use of Compositions Administration of compositions The dose sizes of the compositions ranged from about 1 ml to about 5 ml, based on the antigen content. For example, it can be used for cannons, patches and the like. The route and device that was selected will depend on the composition of the adjuvant, the antigen, and the s are well known to the skilled artisan.

Use of compositions One of the requirements for any d uvant preparation for commercial use is to establish the stability of the grape during long storage periods. Adjuvant formulations which are stable and stable for at least 18 months are provided in the composition. In one modality, the conditions are stable for approximately 18 monthsThe formulations are stable for between approximately 24 months. In another modality, formulated for approximately 24 months. The procedures of the probes also indicate that the formulations of the components are described, although the adjuvant effect is maintained. It is surprisingly surprising that the adjuvant compositions screened herein demonstrate improvements in stopping with other adjuvant compositions.

The adjuvant compositions described in the invention are useful for producing a desired immune response. They are effective in multiple species. A suitable subject is the one for which the administration of a composition is desired, including mammals and non-mammals, including primates, cattle, companions, laboratory animals, wild animals, captives, eggs, reptiles and fish. Thus, this term includes tation, monkeys, humans, pigs, cattle, sheep, mice, rats, guinea pigs, hamsters, rabbits, felines, canid OS, ducks, other poultry, frogs and lizards.

The adjuvants described in the stre. Can be used. This technology is useful in the control and eradication of the subject population.

The following examples are presented as mutative, but should not be taken as limiting the scope. Many changes, modifications and other uses and applications of this invention will be obvious to those skilled in the art.

EXAMPLES EXAMPLE 1 Quil A / Cholesterol Solutions (QC) Quil A (Superfos) was dissolved in water and a re of 50 mg / ml was prepared. Cholesterol (Fabri Chem Inc.) was dissolved in ethanol a stock solution of 18 mg / ml. The solution was then filtered. EXAMPLE 2 DDA Solutions (D) Ammonium bromide was dissolved from dimethyldioctadecyla Analytical), in ethanol, and a stock solution of 18 was prepared after the DDA stock solution using a 0.2 micron filter.

EXAMPLE 3 Solutions of Quil A / Cholesterol / DDA (QCD) A stock solution of A Quil A / Cholesterol co-1 was prepared at the desired concentrations. A solution A was prepared as in example 2 and slowly added to the mad olesterol solution. The solutions were mixed obtaining the desired concentrations. The pH of the solution was adjusted with NaOH or HCl, and CARBOPOL® was dissolved in deionized water and a 0.75% solution was prepared.

EXAMPLE 5 DDA / CARBOPOL® (PC) solutions.

A stock solution of DDA was prepared as in e and prepared a stock solution of CARBOPOL® at 0.75% as a 4. The solutions were mixed obtaining the concentrations.

EXAMPLE 6 Quil A / Cholesterol / DDA / CARBOPOL® Solutions (QCDC) A stock solution of Quil A / cholesterol / D was drawn. EXAMPLE 7 Bay Solutions R1005® (R) To prepare a stock solution of Bay R1005®, -deoxy-2-L-leucylamino-P-D-glucopyranosyl) -N-octadecyldedecanoi was dissolved in ethanol (60% v / v). Then glacial Tween 2 was added. In one example, 3.49 gm of N- (2-d-ylamino-p-D-glucopyranosyl) -N-octadecyldodecanoylamide was dissolved in 44.6 g / water (60% v / v). This was combined with 1.12 ml of Tween 20 glacial acetic acid.

EXAMPLE 8 Quil A / Cholesterol / DDA / CARBOPOL® / Bay R10 Solutions (QCDCR) EXAMPLE 9 DEAE Dextran Solutions (X) A stock solution of DEAE was prepared by dextran 200 mg / ml DEAE in water. The solution was nailed for approximately 20 minutes at 120 ° Celsius EXAMPLE 10 Solutions of Quil A / Cholesterol / DDA / DEAE (QCDX) A stock solution of Quil A / cholesterol was prepared with Example 3. An erdo stock solution was prepared with Example 9. The solutions were combined in a homogenizer. Mixing the ultrafast mixing procedure using a 1000-fold force is "1. Mixing is done by supplying the solution. EXAMPLE 11 Oil Compositions (O) A mother oil solution was prepared was combined! Drakeol with Tween 85 and Span 85, heating to approximate and then cooling and filtering sterile. This would thus hold the base component of the oil phase for an oil in oil. If the cholesterol and / or the DDA were selected as a collaborating unomodulator for one of these compositions, this mixture is also added prior to filtration, since they are sun oily.

EXAMPLE 12 Compositions of Quil A / Cholesterol / DDA / PEAE / Oil (QCD slowly gave into an oily phase that is mixed EXAMPLE 13 Repair of Immunogenic Compositions or Composition Vaccines To prepare an immunogenic vaccine position composition comprising an antigen and one of those described above, the desired antigen was added to an opiate. The components of the adjuvant were then added or described above. The resulting solution was taken up with the buffer.

EXAMPLE 13a geno / Quil A. A stock solution of DDA was prepared as it was slowly added to the solution of antigen / Quil A / antigen strain / Quil A / cholesterol was homogenized and microfluidization of CARBOPOL® at 0.75% was prepared as in the case of microfluidization, the CARBOPOL solution was added to microfluidized composition and the pH was adjusted with NaOH at a maximum of 6.9 to about 7.5.

EXAMPLE 13b Antigen, Quil A, Cholesterol, PDA. CARBOPOL®. Bav R100 To prepare an immunogenic vaccine position composition comprising an antigen, Quil A, BOPOL® stock, and Bay R1005®, the desired antigen was added as desired. A stock solution of Quil A was prepared as in NaOH or HCl at about 6.9 up to approximately a stock solution of Bay R1005® as in example 7. omponent of Bay R1005® to the aqueous phase after A.

EXAMPLE 13c Antigen, Quil A, Cholesterol, PDA, DEAE Dextran To prepare an immunogenic composition of a vaccine comprising an antigen, Quil A, cholest) EAE dextran, the desired antigen was added to an appropriate buffer. stopped a stock solution of Quil A as in example 1 and only to the antigen solution. The composition was homogenized with a cholesterol stock solution as in Example 1 and with the antigen / Quil A solution during the homogenization EXAMPLE 13d Antigen, Quil A, Cholesterol, PDA, DEAE Dextran. Aceit To prepare an immunogenic vaccine position composition comprising an antigen, Quil A, choleste AE dextran, and oil, the desired or opted antigen was added. Be prepared a stock solution of Quil A as in the ex added slowly to the antigen solution. The compo ogenized. A stock solution of cholesterol was prepared as a mixture and slowly added to the solution of antigen / Quil A ogenization. A stock solution of DDA was prepared as an example 2 and added slowly to the antigen / Quil A / solution before homogenization. A DEAE solution was prepared or in Example 9. During homogenization, the s AE dextran was added. An oil composition or an example was read. EXAMPLE 14 Feline Leukemia Virus (FeLV) vaccines The animals were assigned randomly by grilling using a complete distributed block design. Table 1 shows the design of the study. The blocks are date of birth and litter. The animals were classified by litter and then by litter. Blocks of four were used. Den, the animals were randomly assigned to the treatment. Pa vaccination of the study, two consecutive blocks group of eight animals were combined. The groups of animals are assigned to two rooms in such a way that each room or groups (10 blocks) of animals. Within one group of animals, four cages were randomly assigned to each other in such a way that each classroom contained two animals. The vaccines were prepared for this study as in e except that a stock solution of CARBOPOL® was used ecologically. Prepared LEUKOCELL® 2 (Pfizer, Inc.) propagated groups A, B, and C, in lymphoid cells transformed with viral F se se se se se se se se se se se se se se,,,, se est ,nte ,nte ,nte ,nte ,nte .nte .nte .nte. A total quantity of 100 ml of Vete rigation Product (IVP) containing the feline leukemia virus and 25 aluminum p roxide (ALHYDROGEL®) was prepared. A total of 9 FeLV stock solution of 1,106 x 105 ng / ml was slowly added. The pH was adjusted to 5.9 to 6.1 with 4 N HCl or 18 NaOH. They were added, while stirring, 0.5 ml of a solution / ml of Quil A to the antigen solution. Then, 100% v / v ALHYDROGEL® was added. The composition was stirred for 2 hours at 4 ° C. The H was used between 7.0 7.3 with FeLV mother NaOH at 1.106 x 10 5 ng / ml, a 50.0 mg / ml solution of Quil A was slowly added to the antigen solution. They slowly added 0.39 ml of a cholesterol / ethanol solution i. The composition was homogenized for three minutes at 10. 0 added a total of 0.19 ml of a DDA / ethanol solution of 18.0 mposition while stirring. A tot of 1.5% CARBOPOL® solution was slowly added to 145.0 ml of feline leukemia, Quil A, cholesterol, and DDA. The pH was adjusted 7.3 with 18% NaOH or 1 N HCl, as needed.

TABLE 1 Experimental design Number of Phase of Vaccination Trial Phase upo from and IVPa AnimaDomiento Dose Day Day les Via sis Via Vaccination (mi) Test 37. 5 g of 37, 40, LEUKOCELL® 03 20 0, 21 1.0 SC 42d, 1.0 ON 2 Quil A / 44 AI (OH) b 20 of LEUKOCELL® 2 Reformulated 37, 40, 04 Quil A / 20 0, 21 1.0 SC 42d, 1.0 ON cholesterol / 44 DDA / CARBOPOL®b aVeterinary Research bMixed to contain a relative power compared to the reference frame (Reference Lot of FeLV n ° 12) CSC = Su dDepo-Medrol®: Day 42 (approximately 5.0 mg / kg) muscle eON = Oronasal Quil A - cholesterol = Quil A saponin adjuvant, cholesterol lipid artifacts Are sedatives of TELAZOL® (Fort Dodge Animal Health) of bodily agreement (approximately 5.0 mg / kg) via intramus? to minimize animal stress and to avoid damage to the manipulators during the collection of blood. Blood was collected in serum aration (SST) and processed for serum separation, stored at -20 ° C or colder until assayed.

Placebo vaccines or FeLV vaccines were administered subcutaneously at a dose of 1.0 ml. The first vaccination 0 on day 0 and the second administration of vaccine was brought to c 21. All the animals were observed for approximately the first and second vaccinations for didactic reactions (prick reactions). The observed body temperatures of all the animals were measured panic on days 1 and 2 after the first administration of dose d LV) virulent, strain of Rickard, valued at approximately D50 / ml. The FeLV test material was thawed and wet or moist prior to administration. The animals were reared on days 37, 40, 42, and 44, administering 1.0 ml by the undiluted test route. A needle was loaded with a tuberculin syringe, with the test material. Each kitten was administered c nostril. On day 42, the administration was carried out approximately 5 h after the administration of DEPO-MEDROL®. Each day of testing, a sample of the confirmatory testing material was retained.

After the test, a blood sample was collected (1.0 each animal by venipuncture of the jugular vein, on days 64,, 134, 141, 148, and 155. Sedation doses of T rt Dodge were administered) as described above . The blood was collected from serum SST, it was sprayed for its aration. The virus isolation was carried out using mu ro collected on days -2 and 35. Samples from 127 to 155 were considered to evaluate the efficacy of the FeLV vaccine. The serum of day 127 (week 12), day 134 (week 13), day 14), day 148 (week 15) and day 155 (week were tested for the presence of p27 antigen of FeLV. He was persistently infected if he had three or more results of positive FeLV p27 antigen during days 127 (week (week 16).

The temperatures were analyzed using a linear repeated model, and using treatment comparisons between treatment T01 and treatments T02, T03, and T04 to temporal if the general treatment and / or the treatment by poral effect were significant. Averages of dreds, 95% confidence intervals, minimum and maximum p were calculated. No immediate reactions were observed in any of the treatment groups during the first and second vaccinations. Adverse reactions were recorded in any of the groups treated approximately one hour after the first vaccinations. and second, neither pyrexia (body temperature> 39.5 ° C) nor body temperature < 37.0 ° C) in any of the groups of first and second vaccinations. There are no differences in the average body temperature between groups of any time point (p> 0.08). Injection swelling was not observed in any of the treatment groups after first and second.

The final results of week 12 through week 1 ba indicated that 16 animals per 19 (84%) who received l ebo (group T01) were persistently viraemic for FeLV. 13 every 19 68% in the ru T02 were rot id r Thus, the vaccines administered to the T02, T groups were all safe in kittens at the minimum age and were administered in a two-dose regimen, separated three times. The vaccines administered to these groups were able to significantly reduce the level of persistent viremia. kittens at the minimum age when administered in a regimen, separated by three weeks. There was a statistical reduction in the establishment of persistent viraemia of FeLV groups T02, T03 and T04. Additionally, there was an adistically significant difference between T04 and the other vacu groups). It was surprising and unexpected that vaccines that with novel adjuvant ulation would prove to be more effective than commonly used adjuvant components.

They administered sedative doses of TELAZOL® (Fort Dodge Anim according to body weight (approximately 5.0 mg / kg) intramuscularly in order to minimize animal stress and to avoid manipulators of animals during blood collection. of serum separation and processed to separate ro, all animals were observed daily and observed istraron.

The vaccines were prepared as in example 13 ex a 1.5% CARBOPOL® stock solution was used. Se KOCELL® 2 (Pfizer, Inc.) spreading FeLV, subgroups A, B, lymphoids transformed with FeLV. The viral antigens were injected, combined with a sterile adjuvant to achieve the release, and packaged in liquid form. A volume of IVP containing the feline leukemia virus was prepared at one 10,000 rpm. The composition was microfluidized by means of a chamber of limiting dimension of 200 micrometers to 6 447380) pascals (10,000 (+ 500) psi). While stirring, 10.0 ml of a 1.5% CARBOPOL® solution was added to 290 s of feline leukemia, Quil A, cholesterol, and DDA composition. between 7.0 and 7.3 with 18% NaOH or 1 N HC1, as needed The IVP containing the leukemia virus was prepared RP of 5 in the same way as the IVP with a RP of 2 using the stock solution of FeLV and 447.2 ml of PBS buffer with 0.0 quantities of the other components, remaining the same The IVP containing the leukemia virus was prepared RP of 5 in the same way as the IVP with a RP of 2 using a FeLV stock solution, 355.9 ml of 0.063% PBS buffer with Quil A solution, 0.52 ml of the cholesterol solution, and 0.26 DDA volume The IVP containing the RP leukemirus of 20 was prepared in the same way as the IVP with a RP of .9 ml of the FeLV stock solution and 292.0 ml of 3% buffer, with the amounts of the other components following if more.

To administer a dose of 0.5 ml, 30 containing the feline leukemia virus were prepared at an RP of 5, sterol, DDA, and CARBOPOL® in the following manner. 1.7 ml of a FeLV stock solution (35.8 RP / ml where 1 R i of antigen) was added to 277.7 ml of 0.063% PBS buffer. Once, 0.12 ml of a 50.0 μL solution was slowly added to the antigen solution. Then, a cholesterol / ethanol solution of 18 mg / ml was added slowly. Slowly added 0.17 ml of a solution of DDA / ethanol of 18.0 mg standing while stirring. The composition was homogenized dur IVP was prepared to administer a dose of 1.0 mi to feline leukemia to an RP of 5, Quil A, cholesterol, DDA, and CAR in the same manner as the 0.5 ml dose with the carefully adjusted .

A total amount of 300.0 ml of IVP conte of feline leukemia was prepared at an RP of 10 and CARBOPOL®. 2.1 ml of a stock solution of FeLV (50.0 RP / ml where 1 RP i of antigen) was added to 237.9 ml of 0.063% PBS buffer. The co-homogenized for three minutes at 10,000 rpm. The composition was liquidated by passing through a 200 micrometer diaphragm chamber at 68950000 (+3447380) pascals (1) psi). While stirring, 3.3 ml of a 1.5% ARBOPOL® was slowly added to 96.7 ml of the feline leukemia virus. between 7.0 and 7.3 with 18% NaOH or 1 N HCl, as needed.

Placebo vaccines and FeLV vaccines were administered (TABLE 2 Experimental design Number of Potency Vaccine Route Adjuvant for Relative Vaccination Target animals T01 10 N.A. SC PBS Without Adjuvant T02 10 5 RP is FeLV Quil A - Inactivated Cholesterol DDA - CARBOPOL® T03 10 5 RP SC / 0.5 mi FeLV Quil A - Inactivated Cholesterol DDA - CARBOPOL® T04 10 20 RP is FeLV Quil A - Inactivated Cholesterol DDA - CARBOPOL® T05 10 15 RP is FeLV Quil A - Inactivated Cholesterol DDA - CARBOPOL® T06 10 10 RP is FeLV Quil A - Inactivated Cholesterol DDA - CARBOPOL® pain after the administration of test vaccine q aliza, scratches / pitting and aggressive attempt or escape. He measured the attitude after vaccination (normal or abnormal). Males were observed for approximately one hour after the administration of vaccine on Study Day 0 and Day for the development of adverse systemic reactions. It is documented in / ations. The vaccination sites were palpated, and the site of injection, redness at the injection site, injection inflation, and inflammation size were recorded. Study observations 2, 5, and 9 were made after the first vaccination, and study 2, 5, and 9 after the first vaccination, and the days of e and 32 after the second vaccination. Ervations are documented.

A blood sample (1.0 - 2.0 ml) was collected from the anointing in the vein or ular on Study Day 32 to Results - Safety During the first vaccination (Study Day 0), three treatment group T09 showed immediate hard reactions. During the second vaccination (Study Day 20), one year of treatment T05, four of treatment group T08, and or treatment T09 demonstrated immediate immediate reactions.

During the first vaccination, three animals of T09 showed a secondary vocalization. The ani tan pain in the first vaccination also presents secondary alisation at that time. During the second vaccination of the treatment group T05, four of the treatment group of the treatment group T09 demonstrated a vocalization of animals that presented pain in the second vaccination entangled a secondary vocalization at that time. after the first or second vaccination. Neither are adverse conditions in any of the treatment groups.

Results - Efficacy All the animals were proved to be negative an anoation for e! p27 FeLV antigen from samples sampled on Day 1. It was also proved that all animals were from the exposure for the p27 FeLV antigen from mu ro collected on Day 32.

The final results from week 12 to week 16 exposure (Table 3) indicated that 9 out of 10 animals (90%) and treatment T01 (placebo) were persistently viremic for results from the same period indicated that 6 out of 10 animals (60 po of treatment T02 were protected from exposure vir / this level of rotection was not statistically if nifica were protected from the virulent exposure of FeLV, this ection was statistically significant (p = 0.0001) compared with placebo vaccinated 7 of 10 animals (70 %) in the T06 were protected from the virulent exposure of F l protection was statistically significant (p = 0.0198) with the kittens vaccinated with placebo.10 of 10 animals (100%) in treatment T07 were protected from the virulent exposure level of protection was statistically significant (p = stopped with the kittens vaccinated with placebo 8 out of 10%) in the treatment group T08 were protected from the slow e FeLV, this level of protection was statistically sig 0.0055) compared with kittens vaccinated with placebo. ales (50%) in the treatment group T09 were virulently protected from FeLV; this level of protection was not statistically significant = 0.1409 compared with the vaccinated animals with TABLE 3 Summary of the level of protection Discussion The vaccines used in the treatment groups T02, and T07 demonstrated a satisfactory safety profile during the treatment, since no adverse reactions were observed in that ti or animal in the treatment group T05 showed a reaction in the administration, vocalization secondary and attempt to osition with virulent FeLV. That the vaccine provided at 100% protection is surprising and unexpected animals in that group received 25% and 33% of the dose of animals in groups T04 and T05, respectively. An adjuvant adjuvant described and tested in this document allows a lower dose of antigen to be used, although a protective immune response is completely induced. The vaccines administered to treatment groups T02, T06 and T09 showed some protection efficacy (<80% protection), preventive fraction after exposure with virulent FeLV, and the decrease in vaccine administered to treatment group T02. there was likely to be resence of few responding animals in that group.

EXAMPLE 16 Vaccination in Ovo against Eimeria in Chickens altered A general addition of the state of the art, which aims to vaccinate against Eimeria using, for example, recombinant proteins as antigen and a variety of systems, are described in the following publications, all of which are herein incorporated by reference. reference, com bleciera completely, (1) HS Lillehoj et al. , J. Parisitol, 91 (3), -673; (2) H.S. Lillehoj et al., Avian Diseases, 49 2005, 1 12 - 1 17; Alloul et al., Expert Rev. Vaccines, 5 (1), 2006, p.143-163. Enté refers to the use of novel vaccine compositions of adjuvant components that provide a superior in the context of coccidiosis.

The highly effective adjuvants of the present invention are used in combination with the antigenic material of all the species, including their extracts of purified or parked proteins, or by one or more of their expressed proteins of itos in the life cycle of the protozoa. , which includes without limitation sporulated as not sporulated), sporocysts, it is an area, merozoite, male gamete cells or female preferred. The proteins that are released at significant levels in the oocyst phase are the materials that act as the protein antigen source is partially or completely purified from such protein by conventional means.

Additional examples of Eimeria proteins useful in antigen in the formulation of the present vaccines in-vitro by Karkhanis et al. Infection and Immunity, 1991, p. 9 showing protective antigens, as described in these documents, a mass range between approximately 20 and approximately kDa. Additional examples include protein 3-1 E from Eimeria d i rotein Et 100 or e as the E. tenella layer. besia spp. (babesiosis), and related protopzoans, in general icomplexen that causes these or related disorders.

The effectiveness of the in ovo distribution of vaccines that particular adjuvant subjects are evaluated as follows.

Materials and Procedures 1. materials The recombinant maximal E. protein (from the protein expressed in E. coli and purified on an affinity column.) The preparation of whole cell macromolecules of E. maxim (solubiliz tergil of broken cells) was also used as an antigen, a crude antigen that is licked ?? "In a preferred example, the adju mo has been described in Example 8 above, and is prepared as described in Fig. 41. Therefore, in approximately 10 micrograms of DDA; and approximate me rograms of R1005, all provided in, for example, 20 m In relation to the selection of saponin for use in the umento, the following additional information is instructive. The saponin nest refers to the glycosides derived from eroses of which biological properties have been extensively studied (The Plant Glycosides, Mcllroy, R. J., old et al., London, 1951). The most commonly used saponins in the technique for the production of vaccines are those derived from the llano saponaria molina, Aesculus hippocastanum or Gyophilla strut oce extracts of the bark of Quillaja saponaria molina that have an adjuvant activity, for example Quil A. I also wrote Quil A pure fractions that maintain ad activity are at the same time less toxicity than Quil A, for example also described in Kensil et al. 1991. J. Immunolo vol 1 onina refers to "Quil-A" sold in the United States by the Sergeant.

It should further be understood that the sap extracts may be used as mixtures or individual purified components such as fractions / products including QS-7, QS-17, -21 from Antigenics Company, Massachusetts, United States or crude, purified, fractionated saponins. or similar refined products offered by the Isconova Company of Sweden, the Quil A has at least 85% purity. In other models, A has at least 90%, 91%, 92%, 93%, 94%, 95%, 9, or 99% purity. 2. Embryo vaccination The eggs of Moyers Hatchery, Quakert were purchased for in ovo immunization, then chicken eggs were incubated. Doses of 50 microliters were also among those that can be practiced in the present invention. 3. Chickens As soon as the meat chickens were approximately on day 21-22, they were taken to the laboratory using a disposable chicken transporter (Frederick Packagi aukee, Wl) and the chicks were then housed in the units and fed. and water at will.

The birds were kept in pens for pups with no Eimeria and suspended in separate cages where they were infected with live oocysts maximally and kept until the end of the experimental period. 4. Parasites 5. Eimeria exposure infection Seven-day-old birds were tagged in the birds and all the experimental groups except the crate groups were inoculated by esophageal route with maximal E. using uculation, and then placed in oosi collection cages. 6. Determination of body weight gain The individual body weights of days 0 (uninfected), 6 and 10 days after infection with E. were determined. 7. Determination of faecal oocysts production Animal keepers were instructed to cleanse, and faecal stools were collected. Once cages were placed, each cage was placed for 5 days from day 6 of the infection, faecal materials were collected in rows.

Sucrose has been established in Dr. Lil's laboratory. The total number of oocysts detached per chicken was used using the total numbers / bird = (oocysts count x dilution factor x fecal volume / volume of the counting chamber) / number of birds p 8. Collection of samples Samples were collected on day 6 after the date of inf determined the antibody response in serum. Mules of individual birds were obtained (N = 4-5 / group), coagulation was allowed, and serum was collected. Serum samples were assayed for anti-Eimeria antibodies using ELISA. In plates of microtiter plates were rested overnight with the coccidial Ea3-1 E, IC2 recombinant antigens, washed with PBS-0.05% Tween, and blocked with. Serum dilutions were added 1: 20 1: 40 1: 80 1: 1 9. Synthesis of cDNA RNA was extracted from intestinal LELs using TRIzol (I rlsbad, CA). Five micrograms of RNA were treated with 1.0 U of .0 μ? of 10X reaction buffer (Sigma), was incubated during ambient perature, 1.0 μ? of the DNase I stop solution, and the mixture was heated at 70 ° C for 10 ml, inverse the RNA was scribed using the StrataScript strand system (Stratagene, La Jolla, CA) according to the manufacturer's agreement. 10. Quantitative RT-PCR The oligonucleotide primers of the RT-PCR c to the interferon? chicken (IFN-?) and GAPDH control were enumerated. 4. Amplification and detection were carried out using the control panel.

Oligonucleotide binders used for quantitative RT-PCR IFN-v and chicken GAPDH iana of Primer Sequences Size of p PCR RN (bp) APDH Accession No. K01458 264 irect 5'-GGTGGTGCTAAGCGTGTTAT-3 'SEQ ID NO: 1 reverse 5'-ACCTCTGTCATCTCTCCACA-3' SEQ ID NO: 2 FN-Y Accession No Y07922 259 irecto 5'-GCTGACGGTGGACCTATTATT-3 'SEQ ID NO: 3 nverso 5'-GGCTTTGCGCTGGATTC-3' SEQ ID NO: 4 L-? ß Accession number Y 15006 244 irect 5'-TGGGCATCAAGGGCTACA-3 'SEQ ID NO: 5 reverse 5'-TCGGGTTGGTTGGTGATG-3' SEQ ID NO: 6 L-15 Accession No. AF139097 243 irect 5'-TCTGTTCTTCTGTTCTGAGTGATG-3 'SEQ ID NO: 7 reverse 5'-AGTGATTTGCTTCTGTCTTTGGTA-3' SEQ ID NO: 8 The spleen was collected before inoculation with E. ma DPI (date after infection) for the prolife oocyte assay. Spleens were placed in a Petri dish with 10 ml of balanced Hank's salt (HBSS) supplemented with 100 U / ml of 02 and 95% air for 48 hr. The proliferation of 2- (2-methoxy-4-nitrofen) was determined monosodium (WST-8, Cell-Counting Kit-8®, Dojindo Technologies, Gaithersburg, MD). Optical Density (DO) I was using a microplate spectrophotometer (BioRad, Richmond, Results The results showed that broilers and 100 microliters of adjuvant formulation (ie, 100 micro-protein recombinant 3-1 E according to the dose per-dose) gained approximately 45 to 85 grams additional pore compared to unvaccinated birds. but infected x / ma.

The vaccines of the invention also showed effect on cell-mediated immunity as measured by vaccine adjuvant of the invention. In summary, these clearly show the effect of the present adjuvant on the respirators and confirm their effect on the enhancement of the response by cells rather than the humoral response.

The vaccines of the invention also clearly showed the production of fecal oocysts. The control birds without i inaban oocysts. After infection with maximal E., significant losses in the production of fecal oocysts were treated with Pfizer adjuvants alone. Vaccinated birds with maximal gross and adjuvant Eimeria demonstrated a much lower fecal production compared to groups inoculated to preparation of gross maximum Eimeria alone. EM groups.

It should be noted that although purified recombinant protein 3-1 E has been used in the practice of those discussed above, the use of the antigens Ea3-1 E, EaMIF EXAMPLE 17 Bacterin valuation of Escherichia coli strain J5 in g The objective of the study is to evaluate the immune-mediated antigen response of Escherichia coli (strain J-5) when new formulations are added. The commercial J5 bacterin was co-or a preventive vaccine for coliform mastitis in cattle moderately effective in its current formulation. Before the vac, it was determined that the animals had a low antibody titer. coli, based respectively on an analysis of guinea pig samples taken before vaccination.

Cattle Experimental vaccines were formulated using inactivated bacteri oli as an antigen and were carried out according to the protocol.

Vaccine Groups - Cattle Cattle upo No. of Animals Treatment Dose Day (Trat. 01 Saline solution 0, 21 5.0 Escherichia bacterium 02 0, 21 5.0 coli, strain J-5 03 QCDCR 0, 21 5.0 04 QCDO 0, 21 5.0 05 QCDX 0, 21 5.0 06 QCDXO 0, 21 5.0 In table 5, QC is the abbreviation for QuilA / cole DDA, C for carbopol, R for R1005, X for DEAE-dextran ite Mother solutions were prepared as in the examples for the following: E. coli was administered approximately organisms per dose as determined by dir count optical roscopy Quil A in water at 50 mg / ml, cholesterol in eta ml, DDA in ethanol at 17 mg / ml, R1005 in phosphate buffer 20 with Span 80 and Tween 80 (QCDO) or Span 85 and Tween 85 (eol® is a light mineral oil commercially available.

Blood samples were collected on the days of study or serological test. The antibody titers with oli were determined in serum samples by means of a J5 ecologic ELISA assay. Urogen antibody isotypes of anti-bovine sheep antibody (Bethyl Labs) were determined. They stopped and expressed themselves as their geometric means.

Results The serological results of the study are shown in Figures 6-8. Higher antibody titers are generated with better vaccine protection. The specific IgG titer is shown in Table 6. Several of the formulations of the ntion produced much higher titers than the product c TABLE 6 IgG antibody titers The specific lgG1 antibody isotypes were determined. These results are shown in Table 7. Again, Formul O, QCDX and QCDXO were especially effective in inducing a naive immune response in these cattle. These formulations gave higher ho even with a single vaccination than the vaccine injections.

The IgG2 antibody titers shown in the table are frequently associated with better phagocytosis trophiles in milk and protection for the animal. The DX and QCDXO formulations were especially effective in inducing an immune challenge in these cattle.

TABLE 8 IgG2 antibody titers Trat geometric treatment group Title of lgG2 against J5 a day 0 21 48 T01 Saline solution 199 396 396 Escherichia bacterium T02 280 855 3136 coii T03 QCDCR 1 1 14 1402 4966 T04 QCDO 558 1573 6250 T05 QCDX 176 3947 9899 T06 QCDXO 559 8824 87940 s vaccinations were administered by subcutaneous injection studio 0 and 21. The dosage volume was 5 ml.

TABLE 9 Groups of vaccines - Cattle of milk upo No. of animals Treatment _. , T; . Dose Day [ 01 7 Saline solution 0, 21 5.0"" Escherichia bacterium n 02 7 t \ a 0, 21 5.0 co //, strain J-5 03 7 QCDCR 0, 21 5.0 04 7 QCDO 0, 21 5.0 05 7 TXO 0, 21 5.0 In Table 9, QC is the abbreviation for QuilA / cholester, C for carbopol, R for R1005, X for DEAE-dextran, T for LR (CpG-ODN) and O for oil. Solutions were prepared madr iente; E. coli was administered as approximately 4-5 X 109 org dose as determined by direct counting by microscopy mineral oil Drakeol 5 LT with Span 80 and Tween 80 (TXO, n 85 and Tween 85.

Blood collection Blood samples were collected on the days of study or serological test. The antibody titers with oli were determined in serum samples by means of a J5 ecologic ELISA assay. Antibody isotypes of anti-bovine sheep antibody (Bethyl Labs) were determined. He detects titles and expressed himself as his geometric means.

Results The serological results of the study are shown in The highest antibody titers are generally associated with vaccine protection. The total J5 specific IgG titer was plotted.

IgG antibody titers The lgG1 antibody isotypes were determined. These results are shown in Table 10. From QCDO, TXO, and QCDXO nutrations were especially effective for a good immune response in these gains providing much higher titers even with anolation than the commercial vaccine with two injections This isotype of antibody is frequently associated c EXAMPLE 18 Vaccine of bovine viral diarrhea virus Objective of study This study compared the safety, efficacy and safety of two bovine viral diarrhea virus vaccines 2 (BVDV-1 and BVDV-2 or BVD-1/2) and a B-extract vaccine with adjuvants of the invention with a negative control ina) and two positive (a live modified BVDV-2 vaccine and a BVDV-1/2 erta currently available) versus a DV-1 exposure in untreated calves. Table 11 presents the udio.

This study also showed that adjuvants can be used to distinguish vaccine animals. Animal vaccine options of the present invention. TABLE 11 Study design QC is the abbreviation for QuilA / cholesterol, D for DDA, bopol®, R for Bay R1005® received only one vaccination (Study Day 0). They received a modified live V S (MLV) BVDV-2 that did not contain adjuvant. He received a BVDV-1/2 killed virus vaccine containing one in 2.5% water (Amphigen) and Quil A / cholesterol adjuvants. The T04 group received a BVDV-1/2 killed virus vaccine that l A / cholesterol, DDA and Carbopol. Group T05 received a BVDV-1/2 rto vaccine containing Quil A / cholesterol, DDA, Carbopol and? T06 received a high-titre virus extract vaccine containing Quil A cholesterol, DDA and Carbopol on day 0 and a similar low-dose act on day 21. All treatments were administered subcutaneously in a single dose of 2 ml. I gave them the exception of Group 2.

The QCDC +/- R contained 100 pg Quil A, 100 pg of Cole DDA and Carbopol at 0.075% and included 1,000 pg of R1005 per dose or has been described above.

Observations Observations of the injection site study 0 (prevaccination), 1, 2, 3, 7 and 21 for the first site of the left eye were recorded). Observations were recorded for segund ction (also left neck) on study days 21 (prevac 23, 24, 28 and 35) All site reactions were measured (L x A x A, cm). the rectal temperatures gave it -1, 0 (pre-vaccination), 1, 2 and 3 for the vaccination pri straron the temperatures for the reinforcement vaccination gave them 20, 21 (pre-vaccination), 22, 23 and 24.

Collection of Blood Samples Blood samples were collected from each animal by means of serum separation tubes (SST) on study days -1, blood samples were collected using tubes with EDTA what the adjuvants of the invention provided an aur titers against both BVDV- 1 as BVDV-2 as it occurred. An acceptable title for the UDSA is above a tít data shows titles above 5,000 that indicate u ucción of antibodies that is able to stop the alive virus that the animal with potential for infection and disease.

TABLE 12 Neutralizing antibody titre in serum (vaccine, BVDV-1 BVDV-2 adjuvant) Day -1 Day 21 Day 41 Day 56 Day -1 Day 21 Day 41 Average MMCG M CG MMCG Measured MMCG * MMCG * O T01 1. 00 1.00 1.00 21.38 1.00 1.01 1.01 saline, one) (-D (1-1) (1-1) (11-54) (1-1) (1-1) (1-1) OT02 V-2 MLV, roo 1.00 2.75 13.27 1.00 2.14 45.21 one) (1-1) (1-1) (1-27) (1-45) (1-1) (1-10) (1-1024) or T03 35.59? ,? 877.75 1. 00 1.60 486.49 1.00 V-1/2 Pre nt- - sition. A measure of leukopenia is a criterion for the marketing of an MLV product by the USDA. However, inactivated leukopenia is not a criterion for the USDA but in the data the adjuvants of the invention produced only 20% of the animals while the majority of the inactivated irus have a 100% leukopenia. This indicates vantes of the invention were able to direct a T response an inactivated antigen. This is difficult to accomplish and rarely inactivated.

TABLE 13 Leukopenia by Study Day Study day T01 T02 T03 T04 T05 1 Live Solution Prezent QCDC QCDCR QC saline Modified To ExlrÃa 43 0 0 0 0 0 ía 44 0 0 0 0 0 the exact virus in the vaccine. This is observed in that the G stra protection only against BVDV-2. However, vaccines in adjuvant of T03 (PreZent-A), T04 (QCDC) and T05 (QCDCR) g strong antibody response soon to the beginning of the immuni the life phase of the animal study against a serological panel of BVDV. This shows that these adjuvants had the ability to provide safety and efficacy in an exposure model for use not only in a homologous but heterologous exposure.

TABLE 14 Neutralization Certificates in Serum on Day 41 Crossed by The Group Serological Treatment, Log 2 of or Antibody T01 T02 T03 Live Solution Prezent T04 T05 Saline Modified A QCDC QCD V1 to Average < 1 0.8 4.0 2.5 2.9 adora is demonstrated by gel processing by immunoprecipitation (Figure 1). An antibody response of NS2 / 3 and E2 antibodies of BVDV is very pronounced in an animal and an MLV vaccine or an animal naturally exposed to na inactivated with PreZent-A adjuvant. However, the adjuvant antibodies showed only an E2 antibody response and not against the NS2 / 3 proteins. Therefore, an animal and an inactivated BVDV vaccine comprising adjuvant can be differentiated from an animal naturally infected with the one vaccinated with MLV or animal vaccinated with PreZent-A. I would devise a marker vaccine that is valuable for the eradication of types of diseases in animal populations.

EXAMPLE 19 Mycoplasma hyopneumonia in Suidos The MPS causes considerable economic losses in farms where pigs are reared. Surveys carried out in lizations all over the world indicate that the typical lesions of lMvan with MPS appear in 30% -80% of the pigs in the hole. Because the mycoplasmal lesions can resolve that the pigs reach the slaughter weight, the actual incidence does. It has been described that the prevalence of pneumoniae infection in chronic pneumonia of swine ranges from 2 to 2 of all ages are susceptible to MPS, but the disease is common in growing and finishing pigs. Tests indicate that M. hyopneumoniae is transmitted by aerosol or contact secretions from the respiratory tract of infected pigs. It is the sowing of sows to piglets during lactation. Once this S appears year after year in infected herds, varying in environmental grav ores such as season, ventilation and concentration d Animals Sixty-six (66) healthy pigs at about 17 days of age were used in the study without a disease caused by M. hyopneumoniae and PRRSV or with the same organisms. Before transport to the site for 2 days after arrival, the pigs were treated with intramuscular Nax on the hind leg, following the instructions to prevent diseases related to stress, ptococcus suis. The animals were assigned to treatments and are under a randomization plan. The study design was shown 15.

TABLE 15 Experimental design No.

Vía Vaccination Treatment Veterinary Research Products (IVP) The Antigens and Veterinary Research Products are listed in Table 16. Vaccines for the treatment groups, and T04 (all except T05) were prepared according to the following concentrations of components shown below. The components were added in the order in which they were added.

A plasma expander of saline solution was added and homogenization was initiated and continued during the preparation run. M. hyopneumoniae was prepared from a mixed volume of 75 liters fermented by 800 formulated final ducts and added at a concentration of 0.093 is. Quil A was added at the concentration listed in which cholesterol / ethanol solution was added. Ethanol was added followed by addition of the glycolipid solution Ba TABLE 16 Veterinary Research Products (IVP) Vaccination The animals in the NTX treatment group were not vaccinated. At approximately 3 weeks of age (Day 0 a 1: 50 dilution in Friis medium of a 10% gelled homogenate of M. hyo strain 11 (LI36).

Collection of Blood Samples Day -1 or 0 (before the 1st vaccination), Day 13 or 14 2nd vaccination), Day 34 or 35 (before the exposure) and Day ropsia), blood samples were collected (approximately 5 serum separators) ) of all the pigs and the ology of M. hyopneumoniae (ELISA-IDEXX) was tested.

Weight All animals were weighed on arrival with tribute in batches, on Day 34 or 35 (before the exposure) and the Day of the necropsy). a lobe (left cranial, left half, left caudal, cho, right middle, right caudal, and accessory were scored r real between 0-100%.) The percentage for each pulmonary lobe s weighted formula for the calculation of the total percentage of pulses. A necropsy of six (6) NTX animals was performed on Day s of exposure and their lungs were scored for lesions.

Pulmonary Injury Scores The percentage of total lung with lesions was calculated as a formula: Percentage of total lung with lesions = 100 x left eal) + (0.10 x left half) + (0.25 x left flow 0 x right cranial) + (0.10 x right half) + (0.25 x flow d 0 x accessory)} . The square root transformation of the total lung arch with lesions was applied before analysis. The transformed nonares were analyzed with a linear mixed model Results As indicated by the results of table 17 below adjuvants of the invention behaved just as well as the with adjuvant oil T05 containing the a phigen®. Typically, with a lung injury score p it is considered that efficacy has been conferred by the treatment of combinations of the adjuvants of the invention meet these CDCR behaved the best in score and interval between viduales.

Table 17. Percent of Lung with Injuries Signal Ratio: Positive serological MMC Interval (S / P) Day 34 T01 - Placebo 0.00 8.4 0-25.55 T02 - DRC 0.28 2.4 0-20.13 T03 - QCDC 0.15 2.1 0-23.18 EXAMPLE 20 Feline Avian Influenza Virus (FAIV) This study evaluated the efficacy in cats of a strain using an adjuvant of the invention by exposure to an S of virulent avian influenza.

Procedures and Results Prior to the vaccination, it was determined that the anim als for both influenza and co-antibody antibodies, based respectively on oropharyngeal swabs and blood serum samples taken before vaccination.

Experimental vaccines were formulated using inactivated gen and purified haemagglutinin (HA). Each group initially contained six animals (table 18). Two times until the animals recovered and were taken down to make sure that there were no reactions adveted the observations at about one hour post-vac recorded any other complication observed afterwards.

The adjuvant composition has been described previously by the QCDC example using Quil A (20 pg), C pg), DDA (10 pg) and Carbopol (0.05%) per dose. The inactivated pleo antigen or purified HA H5 protein.

The animals were evaluated for reactions in the ction and serological response to the vaccine. Three were inactivated in T02-H5N2 inactivated and one in T05-saline solution) d congenital roxaluria before exposure. On study day 49, surviving t S were exposed by the intratracheal route to ce etnam / 1194/04 to evaluate the efficacy of the candidates for vaccination. TABLE 18 Vaccine Groups Blood samples were collected on the days of minting, 0, 21 and 49 for serological testing. The study days echoed blood samples for virological testing. On day d, an unscheduled blood sample of all characteristics attributable to a FAIV infection was taken. S ettings to identify the degree of lung consolidation. The residue was fixed with 10% neutral buffered formalin for right-side histop was collected and samples were obtained for logic. In addition to the lungs, a sample of any tissue with macroscopic pathology was also taken and stored in a 10% neutral dose for histopathology.

Viral titres were determined in post-oropharyngeal and rectal samples and in lung tissue samples using a TaqMan specific H5N1 PCR. In summary, the MagnaPure LC isotope was isolated with the nucl naPure LC acid isolation kit (Roche Diagnostics, Almere, The Netherlands), and influenza A by using an RT-PCR assay at time s were expressed as Control dilution units (UUs were run from a standard curve produced in parti) Plasma samples were analyzed by neutralization of hemagglutination inhibition.For the inhibition of agglutination (Hl) test, a suspension of virus was incubated. enza Vietnam 1 194/04 (H5N1, class 1) or Indonesia 05/2005 (H5 on serial dilutions (2 times) of pretreated serum sample of cholera (obtained from cultures of Vibrio cholerae), later erythrocytes at dilutions and after incubation, the number of agents exhibiting complete agglutination inhibition was defined as the Hl titer.

The virus neutralization (VN) assay was based on the endpoint of the sera. In summary, one tantalum of virus was mixed with a serial dilution (2 times) of a mu. Virus neutralization was read using commercial MDCK cells and visualized by agglutination of erythrocytes. Score VN taking the highest dilution of serum to which the 50 Results None of the animals in the five groups experienced pain or swelling at the injection site after the single vaccination. In addition, there were no anomalies in the injection pi s. After the vaccinations and before the expo ia significant differences at the level of significance of 0.1 corporal peratures between treatments by mod al analysis. An animal of T01 was febrile (> 40 ° C) before the first going 0 and for several days afterwards. Increases were recorded in body temperature in individual animals up to 40 ° C or after vaccinations (Days 0 and 21). No salutation was observed with vaccination during the study. Three animals (do 2, and one in T05-saline) were euthanized due to hip genitia before exposure. Several animals of all treated wounds complications after the implant s the animals remained healthy after the exposure. The first abnormal clinical signs (depression and receding effort) were observed in two animals two days later. Three days after the exposure, the six animals were depressed and showed an increase in respiratory effort, two animals had to be euthanized for reason. Four days after the exposure (Day 53), the dead were placed and the remaining three animals of T04 presented with increased respiratory distress, protrusion of the third eyelid and al and were euthanized for reasons of well-being. At T05 (n = 5), abnormal clinical symptoms of depression and respiratory effort increased in one animal one day after exposure. Two days of exposure, two more animals began to show these signs after the exposure, a dead animal was found, and the remaining animals had depression, increased respiratory effort. The mean temperatures of the control animals (T04 and aron up to 40.0 ° C, beginning one day after the exposure in average body temperatures between treatments (p = 0.0001) by linear mixed model analysis. in a minority of animals and T03, body temperatures rose to 40.0 ° ma at sporadic time points on Day 53. In T01, two febrile bans (range 40.0 to 40.1 ° C) at one point of time. febrile (range of 40.0 to 40.3 ° C) in one of the time zones, respectively In T03, one of the animals (range 40.0 to 40.3 ° C) at three time points In TO> s the animals were febrile for at least twelve hours 50 and 51.

Antibody titers of Hl against the strains of i nam 1 194/04 (H5N1, class 1) and Indonesia 05/2005 (H5N1, claws of the second vaccination.) In T02, the titers against / 04 were lower than those observed in T01 and T03, ranging from the first vaccination and from 5 to 70 after the nation, the antibody titers of Hl against Indonesia 05/20 to those observed against Vietnam 1194/04.

Plasma samples taken before and exposed by H5 specific real-time RT-PCR were assayed for viral load undermining. All animals had neg samples before exposure. After exposure, no animal asthma of T01 and T03 was detected. On the contrary, e! 25% (1 of T02 ales, 67% (4 of 6) of the T04 and 60% animals (3 T05 ales were positive to virus in plasma after the exposure differences between treatments (p = 0.0247) per year). mixed linear.

The elimination of virus after exposure was evaluated by taking samples of animals from T04 and T05 five days after, since all the animals had died for a statistical analysis, however, the results of the last animals that died or They were euthanized before the end of the eronon on the last day of the study.

Throat specimens were also used with an RT-PCR (> 1.8 UDC) assay in virus-lation titration assays confirming that all positive samples had infectious influenza virus (data not shown). The infectious S were lower in vaccinated animals (T02 and T03 control diseases (T04 and T05) .These differences were significant >after the exposure when compared to T02 or T03 c > , four and five days after exposure when comparing 03 with T05. Animal titers of T02 and T03 were 0.5 log and titres observed in T04 ranged from 2.3 to 4.3 log10 TCID50. L Pulmonary pathology was less severe in animals, T02 and T03) than in control animals (T04 and T05). All patients had a subacute bronchointerstitial pneumonia. The control animals showed moderate bronchoin- gonae pneumonia (two animals of T04 and one animal of T05) of animals of T04 and four of T05) with a multi-s distribution, except for two control animals. they showed (an animal d of T05) a diffuse distribution. The lung was evaluated to complete the degree of consolidation, which was expressed in percent solidation of total lung tissue. According to the lung logicians, the percentage of consolidation was significant in vaccinated animals (T01, T02 and T03) than in animals d and T05).

The viral load in lung tissue collected from death or euthanasia was evaluated by virus titre and lung RT-PCR and pulmonary pathology including consolidation, in animals that had received adjuvant (T04) or saline solution (T05).

Vaccination with purified HA H5 protein (T01) pria, viral elimination from the throat and young mortality after exposure with a pathogenic avian influenza strain. In addition, vaccination with HA protein H5 p) reduced clinical signs including fever, viral load in the lung, including consolidation.

Vaccination with an inactivated H5N2 strain (T02), clinical pre- disposition, viral elimination in feces and mortality in cuts after exposure to a pathogenic avian influenza strain. In addition, vaccination with the H5N2 in) strain reduced viraemia, fever, viral clearance from lung gargana and pulmonary pathology, including consolidation.

Vaccination with an inactivated H5N1 strain (T03) pre Summary No nasal injection site reactions formulated with HA antigen inactivated or purified with DC were observed. The vaccines provided complete protection against fever and mortality in vaccinated cats, significant viral load in blood and tissues, and significant virus clearance.

EXAMPLE 21 Cancer Background This study was conducted in one-competent immunodeficient rats using hum hepatocellular carcinoma cells to generate heterotopic and orthotopic models. ximately 7 weeks and euthanized by COXperiment inhalation.

The experimental design incorporated two phases. They randomly rats the rats into two groups based on oral. The rats in group 1 did not receive t cell injection after the rats in group 2 received a subcutaneous injection of the tumors. Three weeks after the injection of t S into group 2 were randomly distributed (based on the ral and the intake index - see table 19) into two group II, including one of the same two subgroups: 1) control The tumor was treated with a saline solution (the Group of Co-roles that had tumors treated with adjuvant only (Tumor), and 3) subjects who carried tumors (Tumors ficados with vaccine (two subcutaneous injections separated anas). animals to TABLE 19 Vaccine groups Types Number by Cells N ° of Formulation Description Tumor Vaccinations Animals (Antigen) Placebo de Role Without Negative Control NA PBS at mor (saline solution) 0. 63% Placebo de Vehicle without QCDC PBS at ict) antigen 0. 63% Liquid dose QCDCR and Placebo de homogenized mor- 100 μg of PBS at tado) vehicle plus antigen 0. 63% HepG2 inactivated QC is the abbreviation for QuilA / cholesterol, D for DDA, opol®, R for Bay R1005®, PBS for saline buffer Vaccine Preparation Vaccines were prepared using Quil-A (20 cg / dose), DDA (10 g / dose), Carbopol (0.05%) with or without 15-year-old Cassian glycolyol with a hepatocell carcinoma renciado. The cells were expanded under culture conditions and prepared for injection at a concentration of cells / ml in Matrigel. Each rat was injected 0.5 ml of its lar, subcutaneously at the site of the second nipple.

Measurements The tumor size was measured twice a week during the caliper procedure, where the volume (A (mm) x A (mm)] / 2 x L (mm). / 1000. Blood was collected per orbital The chemical and serum biomarker measurements were slightly anesthetized during the CO2 / O2 procedure, and the chemical endpoints or analyzer Hitachi 917 (Roche, Indianapolis, IN) were analyzed under anesthesia with C02 by cardiac puncture. The values were expressed as mean ± SD and a p-value that was statistically significant.

Results Body weight measurements were corrected by subtracting ral (based on the volume data and on the assumption that the data was analyzed in two ways: by group of patients with or without tumor. there was a significant difference between the groups in terminal time, there was no difference in the basal level, even if the body was not significantly different when co-treatment group, probably due to the short-term halt, there was an appreciable tendency towards a body weight if there were groups that carried a tumor with respect to the controls and a TABLE 20 Change in body weight with time between rimentales. The shaded areas indicate data when it is added.

Body Weight (g) Control Tumor Tumor Treated 0 242.7 ± 1 1.4 260.0 ± 16.3 245.0 ± 12.1 6 263.9 ± 13.5 278.1 ± 17.5 258.6 ± 14.6 9 270.1 ± 14.0 280.6 ± 19.2 260.1 ± 15.1 13 280.6 ± 16.2 290.3 ± 20.2 262.0 ± 15.1 16 283.8 ± 15.0 289.0 ± 18.5 261.1 ± 15.3 20 292.6 ± 16.1 284.1 ± 17.3 259.2 ± 14.6 23 299.8 ± 15.6 283.2 ± 17.0 252.7 ± 14.2 27 298.7 ± 14.0 276.1 ± 15.5 259.4 ± 14.4 30 307.6 ± 15.9 274.2 ± 3.4 260.9 ± 14.5 34 317.8 ± 16.2 262.6 ± 14.6 267.8 ± 15.1 37 318.1 + 16.6 263.1 ± 15.0 268.4 + 14.8 41 323.4 ± 15.3 264.4 + 14.7 274.9 ± 15.2 44 328.5 ± 16.6 262.5 ± 14.6 278.3 ± 15.1 48 331.2 ± 17.0 263.0 ± 14.1 281.6 ± 15.0 49 330.5 17.1 262.6 ± 14.4 281.2 ± 14.3 Tumor Size TABLE 21 Change in tumor size between the group treated with or) and the group treated with vaccine (Tumor treated). The breadas indicate the data of when the vaccine was administered.

Tumor Size (cm3) Tumor Tumor Treated Day 6 4.6 ± 2.1 4.7 ± 2.0 9 4.9 ± 2.0 4.8 ± 1.8 13 4.6 ± 1.8 5.0 + 2.2 16 12.2 ± 2.7 12.3 ± 2.5 20 22.6 + 3.8 13.4 ± 2.6 23 33.3 ± 4.8 14.1 ± 2.8 27 34.5 ± 4.6 13.8 ± 2.8 30 35.6 ± 4.6 13.5 ± 3.4 34 37.7 ± 8.0 14.1 ± 2.9 37 38.6 ± 10.2 13.7 ± 2.3 41 44.5 ± 12.1 12.5 ± 2.1 44 52.9 ± 13.9 13.0 ± 2.1 48 61.7 ± 15.1 16.9 + 2.9 TABLE 22 ales in treatment groups. The data from this study and the rich indicate that AFP is only detectable in animals that carry the longitudinal data of AFP in the control and treated groups indicates that the AFP decreased in the animals of the first injection of vaccine and was much less than the controls that carry tumor at the end of the study; 4. in vehicle treated with respect to 0.97 ± 2.5 ng / ml in vaccine rats, respectively. In addition, the AFP demonstrated a co with the tumor volume as with the tumor weight removed.

Human albumin (hALB) was measured by ELISA at time during the study. The data from this study and the rich indicate that hALB is only detectable in animals. The comparison of the hALB data in the treated and treated groups indicates that the hALB was lower in treated rats that carry tumor at the end of the study (the lesion is not shown and by animals that carry or do not carry a tumor. The only valuations in which differences were observed were: AST sterol For both comparisons, there were no sign differences in the basal time point (the data are not shown), they compared the chemical indices in animals that carried your ales that did not carry tumor, there was a significant difference in the terminal time point with elevations in AS sterol in the animals bearing the tumor (datum not shown). conclusion As a whole, the data show that the burden was on animals treated with the vaccine prepared against the oral line HepG2 with respect to a group that carried coba vehicle tumors.

Materials and Procedures Female C57BI / 6 mice (n o) with a body weight of approximately 18-20 g were used in the study. Is intramuscular immunization (IM) in the left anterior tibial muscle total of 50 μ? the study days 0, 14 and 21.

Reagent Dosage A dose of the composition comprised, in binations, one or more of the following components: Buffer: NaH2P04.2H20 (229.32 mg / l), NaCl (1168.0? 04 (1144.00 mg / l), dissolved in WFI and sterilized by 0.1 μ filtration.

Ovalbumin (OVA-Antigen): 10 μg CpG ODN: 10 μ Vaccine Preparation Buffer was placed in a 50 ml flask with one portion and stirred at a constant speed during all the steps. The components were added in the following order: / A); CPG ODN; Quil A; cholesterol (drop by drop); DDA (drop bopol®; and Bay R1005®.) The composition was stirred at a temperature of 25 ° C at a temperature of 30 ° C for a minimum of 30 minutes, coated with aluminum foil. The solution is from a 25G needle in a syringe to break any large size to obtain a uniform suspension (it was transferred to sterile glass vials for storage.

Collection of samples The following samples were collected: Plasma: 4 weeks after the Tetramer sensi vaccination (4 weeks after the sensitization vaccine) T cells producing cytokines (6 weeks after sensitization) The results are given as a score each adjuvant showing the effect of the adjuvant. The cri-ration was a relative scale based on the sum of the individual cytotoxic T responses.

Results and Discussion As presented in Table 23, QCDCR triggered stronger CTL responses than its subcomponents argo the set responses were low (< 20%). The combination CR or its subcomponents with CPG significantly improved specific OVA CTLs. QCDCR / CPG plus OVA in ncia to increase cellular immune responses. The combination two shows synergy. When sub-components of CpG were analyzed, combinations with Quil A gave the best value of inclusion of cholesterol with CpG.

TABLE 23 CTL Relative Answers EXAMPLE 23 Canine Coronavirus (CCV) Ambit A murine model using canine coronavirus S combination adjuvants was used to evaluate the yield of the given antigenic component.

Animals Ten CF-1 mice per treatment group were administered subcutaneously per animal of each treatment group.

Treatment Groups The test formulations shown in c were repaired as field dose volumes of 1.0 ml. TABLE 24 Test Formulations.

Vaccine Preparation The vaccine preparation for the adjuvants of i in the above examples 1-13. The concentration 0000 pascals (10,000 psi). Then Carbopol was added with the pH at 6.8 to 7.2. Glycolipid Bay R10 a. Finally, the composition was brought to a final volume with the saline solution attic.

The vaccine for the treatment groups that AbISCO products available in the market (Isconova, Su plowed according to the instructions of the label.) The prod CO are based on Quillay saponins and the technology of highly purified saponins.

Test Procedure: The serum neutralization of C The serum was inactivated by heat at 56 ° C for 30 to 40 min sterile clean plate were serial dilutions of each s, 2, 4, 8 ...) passing 120 μ? at 120 μ? of diluent. The wells / dilution measurements were used. A dilution d 2 days before was initially used. The CPE was evaluated 4 to 6 days later. The retro He claimed that 50 to 316 virus particles affected every single Results TABLE 25 Neutralization Sérica Group of Neutralizing Titles in Serum treatment Saline solution 2 Antigen only 64 AblSCO-100 256 AblSCO-200 23 AblSCO-300 1 1 Quil-A / Cholesterol 315 R 512 RC 11 DRC 630 QCR 1024 QCDC 630 QCRC 724 QCDRC 1448 Adjuvant formulations of the invention yielded titres that the commercially adjuvanted products, even when they had a similar amount of CCV antigen, QCDC, QCR, DRC, QCRC and QCDRC ulations were especially in the induction of a good immune response in the mouse EXAMPLE 24 Bovine Rotavirus antigen Ambit A murine model using Rotavirus combination uvantes of the invention was used to evaluate the yield with the given antigenic component.

Animals TABLE 26 Test Formulations Vaccine Preparation The vaccine preparation Ethanol was then added during homogenization. The fluidized me to 68950000 paséales (10,000 psi). Then it was mixed with opol® and the pH was adjusted to 6.8 to 7.2. Bay R1005® was added with mixture. Finally, the composition is finalized with the plasma expander of saline.

Vaccine for the treatment groups receiving AbISCO products available on the market (Isconova, Sue aró according to the instructions on the label) The products are based on Quillay saponins and highly purified saponin technology.

Results TABLE 27 Neutralization Titers in Serum Formulations of Neutralizing Titles in QC is the abbreviation for QuilA / cholesterol, D for DDA opol®, R for Bay R1005® The combined effects of the adjuvants formulated Bovine virus and taking into account the chemical properties of the presenter have provided excellent properties for an adjuvant (see Table 27).

Although several of the adjuvant formulations provide similar neutralizing antibody titers in S QCDCR, the highest level was provided.

EXAMPLE 25 Canine Influenza Virus Scope / Study Design A canine model using influenza virus cani sensitivity to vaccines available in the market was used. The animals received vaccines against CIV.

TABLE 28 Study Design QC is the abbreviation for QuilA / cholesterol, D for DDA bopol® TABLE 29 Composition of Vaccine 01 Adjuvant placebo, negative control Vaccine Preparation The vaccine preparation for adjuvants of i describes in the above examples 1-13. The concentrations of adjuvant presenters are given in Table 29. They are added in the order listed in the table.

A plasma expander of salient solution was added and homogenization was initiated. Influent virus was added at a concentration shown in Table 29. S l A at the concentration listed in Table 29. Sterol / ethanol was then added with homogenization containing DDA / ethanol was then added during the homogenization zcla was microfluidized at 68950000 pascals (10,000 psi). Carbopol dioxide was mixed and the pH was adjusted to 6.8 to 7.2. By position it was taken to a final volume with the saline expander.

TABLE 30 HAI Titles The combined effects of the adjuvants formulated enza and taking into account the chemical properties of each co provided excellent properties for a vaccum adjuvant Larger antibody titers are generally associated with vaccine protection. Generally, both the adjuvant of 2) and the adjuvants of the invention (T03, T04, and T05) came under HAI titers but the response caused by the ad the invention was higher with titres greater than day 180 in the group.

Claims (1)

1. NOVELTY OF THE INVENTION CLAIMS 1. - An immunogenic composition comprising an adjuvant and an immunologically effective antigenic amount, wherein the adjuvant formulation comprises aponin, a sterol, a quaternary ammonium compound and a pol 2. - The immunogenic composition according to disclosure 1, further characterized in that the saponin is pres eated from about 1 μ9 to about 5,000, the sterol being present in an amount of about im mimately 5,000 μg per dose, the ammonium compound present in an amount of about 1 μg to about μg per dose and the polymer is present in a quantity A. The immunogenic composition according to disclosure 1, further characterized by comprising additional Th2. 5. - The immunogenic composition according to disclosure 4, further characterized by the Th lipid stimulant. 6. - The immunogenic composition in accordance with disclosure 5, further characterized in that the stimulant of to of A / - (2-deoxy-2-L-Ieucylamino ^ -D-glucopyro ecildodecanamide. 7 -. 7 - The immunogenic composition in accordance with 5t disclosure further characterized in that the glycolipid is presorbed from about 0.01 mg to about 10. 8. - The immunogenic composition according to the position of step c; e) adding the polymer to the composition of 10. The process according to claim 1 further characterized in that the saponin is Quil A or a fraction p itself, the sterol is cholesterol, the ammonium compound quate and the polymer is polyacrylic acid. eleven . - The method according to claim 1 further characterized in that it additionally comprises an ogenization of the composition of stage a and the continuation during each of stages a to d. 12. - The method according to claim 1 further characterized by additionally comprising a microfluidized ethereal composition of stage d. 13. - The method according to claim 1 further characterized in that it additionally comprises an antigenic eta ponent, in which the adjuvant formulation co saponin, a sterol, a quaternary ammonium compound and a p 17. - The composition of vaccine according to indication 16, also characterized by the saponin. is pre-amount of about 1 μ9 to about 5.00 is, the sterol is present in an amount of about 5,000 μg per dose, the ammonium compound cu present in an amount of about 1 μg to about 0 μg per dose and the polymer is present at a maximum of 0.0001% v / v approximately 75% v / v. 18. - The vaccine composition according to indication 16, further characterized in that the saponin is purified from it, the sterol is cholesterol, the quaternary compound is DDA and the polymer is polyacrylic acid. 19. - The vaccine composition according to A / - (2-deoxy-2-L-leucylamino ^ -D-glucopyra decyldodecanamide. 22. - The vaccine composition in accordance with specification 20, further characterized in that the glycolipid is pre-amount of about 0.01 mg to about 1 is. 23. - The composition of vaccine according to ndication 16, characterized in that said component also ignites an inactivated virus. 24. - A method of preparing a composition as claimed in claim 16, which comprises adding a composition of the antigen component in a buffer; aponinates the composition of stage a; c) adding the position of stage b; d) adding the ammonium compound quater position of step c; e) adding the polymer to the ogenization composition of the composition of step a and the continuation during each of steps a to d. 27. - The method according to claim 1 further characterized in that it additionally comprises a method to microfluidize the composition of step d. 28. - The process according to claim 1 further characterized in that it further comprises an eta to the composition of step e, a Th2 stimulant. 29. - The method according to claim 1 further characterized in that the Th2 stimulant is a glycolipid. 30. - The method according to claim 1 further characterized in that the Th2 stimulant is acetate oxy-2-L-leucylamino- -D-glucopyranosyl) - / V-octadecyldodecanamide. 31. - The immunogenic composition according to indication 1, also characterized in that it comprises additional 34. The method according to any of the ndications 9 to 15 and any of the claims 2 further characterized in that the antigenic component comprises feline leukemia. 35. - The vaccine composition of any of claims 16 to 23, further characterized in that the com- mon comprises the feline leukemia virus. 36. - The vaccine composition according to ndication 35, also characterized by the leuke virus. present in an amount of approximately 100 x 350,000 ng / ml, inclusive. 37. - The immunogenic composition in accordance with ndication 1, further characterized in that the gp70 component produced by a FL-74 cell line persists with the feline leukemia virus strain KT-FeLV-UCD-1. romoléculas isolated from said protozoan by encionales and (3) complete cell extracts or zoo preparations. 40. The immunogenic composition according to claims 1 to 7 and 31, further characterized as antigenic agent comprises a bacterin of the strain erichia col i. 41. The method according to any of the ndications 9 to 15 and any of the claims 2 further characterized because the antigenic component comprises erin of the J-5 strain of Escherichia coli. 42. The vaccine composition according to UC claims 16 to 22 and 32, further characterized as antigenic agent comprises a bacterin of the strain erichia coli. 45. - The immunogenic composition in accordance with ndication 44, further characterized in that the component underlies BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). 46. - The method according to any of the ndications 9 to 15 and any of the claims 2 etherified further because the antigenic component comprises B 47. - The method according to claim 1 also etherified because the antigenic component comprises B V-2. 48. - The vaccine composition according to C claims 16 to 23, further characterized in that the coment comprises BVDV. 49. - The composition of vaccine according to ndication 48, characterized in that the component a catches BVDV-1 and BVDV-2. 52. - The method according to any of the ndications 9 to 15 and any of the claims 2 further cterizado because the antigenic component co opneumonia. 53. - The vaccine composition according to claims 16 to 22, further characterized in that the coenic comprises M.hyopneumonia. 54. - The use of a vaccine composition such as ama in claim 53, in the preparation of a medicament to a suid against an infection caused by M.hyopneumonia. 55. The immunogenic composition according to any of claims 1 to 8, further characterized as an antigenic agent comprises the feline influenza virus (FIV). 56. - The method according to any of the indications 9 to 15 and any of the claims 2 59. The immunogenic composition according to claim 1, further characterized as an antigenic component comprises a cancer antigen. 60. - The method according to any of the ndications 9 to 15 and any of the claims 2 also etherized because the antigenic component comprises cancer. 61. - The vaccine composition according to claims 16 to 22, further characterized in that the comon comprises a cancer antigen. 62. - The use of a vaccine composition as in claim 61, in the preparation of a medicament for a subject against cancer. 63. - The immunogenic composition according to claim 1 to 8, further characterized 66. - The method according to claim 1 further characterized in that the ORN / ODN is CpG. 67. - The vaccine composition according to claim 16, further characterized by an ORN / ODN. 68. - The vaccine composition according to indication 67, also characterized because the ORN / ODN is CpG 69. The immunogenic composition according to any of claims 1 to 8, further characterized as an antigenic agent comprises a canine coronavirus (CCV). 70. - The method according to any of the indications 9 to 15 and any of claims 2 further characterized in that the antigenic component comprises 71. - The vaccine composition according to claims 16 to 23, further characterized in that the 74. The method according to any of the ndications 9 to 15 and any of the claims 2 further characterized because the antigenic component bought bovine virus. 75. - The vaccine composition according to claims 16 to 23, further characterized in that the coenic comprises a bovine rotavirus. 76. - The use of a vaccine composition like the one in claim 75, in the preparation of a drug to a bovine against an infection caused by a bovine rotavirus 77. The immunogenic composition according to any of claims 1 to 8, further characterized as an antigenic agent comprises a canine influenza virus (CIV). 78. - The method according to any of the ndications 9 to 15 and any of the claims 2 81. - A method of differentiating a natural animal with BVDV from an animal vaccinated with a compound as claimed in claim 48 or 49, or obtaining a sample from a test animal and providing them with E2 protein and NS2 / 3 proteins in said sample, in NS2 / 3 protein indicates that the animal was vaccinated c vaccine position.